The principal goal of this R21 application is to determine the source of extracellular glutamate released by primary human glial tumors (gliomas). In normal brain, glial cells avidly transport glutamate, use it to detoxify ammonia by synthesizing glutamine (via glutamine synthetase (GS)), and export glutamine into the extracellular space and blood via specific glutamine transporters. Neurons transport glutamine and convert it back to glutamate via phosphate activated glutaminase (PAG) for synaptic neurotransmission. This glutamate-glutamine cycling is radically altered in gliomas. Instead of clearing extracellular glutamate, gliomas take up glutamine and release ammonia and glutamate through specific co-transporters in sufficiently high concentrations to induce excitotoxic damage to the surrounding tissue. The death of surrounding normal neurons and glia allows the glioma to invade into the surrounding neuropil. However, the specific pathway(s) by which glutamate is synthesized from glutamine in glioma cells are not fully understood. It has been hypothesized that the changes in stoichiometry would allow GS to run in reverse. However, GS is an ATP-dependent enzyme, and while it is highly expressed in some gliomas, thermodynamic and enzyme kinetic considerations suggest that GS is not a source for glutamate production. Our pilot data show that GS activity is dramatically reduced in higher grade tumors. Therefore, we hypothesize that glutamine is converted to glutamate and ammonia by glutaminase expressed by the gliomas. In Specific Aim 1, we will test this hypothesis in two systems: acutely isolated human glioma and human glioma cell lines grown in the mouse cortex. If this hypothesis is confirmed, it would provide a new target for pharmacologic therapy. Western blots and immunohistochemistry will also be done to confirm the functional studies. In Specific Aim 2, we propose that changing the fuel balance to one favoring fatty acids and ketone bodies with reduced glucose/glycogen availability will reduce extracellular glutamate release by increasing glutamate oxidation and thus reducing the ability of tumor cells to proliferate. These studies will be done in both preparations. We will primarily measure glutamate and glutamine metabolism in human and murine tissue using ex vivo slices exposed to 13C labeled compounds. The pattern of isotopic enrichment will be determined using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The strength of this is approach is that it allows us to study both glutamate metabolism but also glutamate efflux in a system that has neurons, astrocytes as well as glioma cells. As part of this project, we will address the metabolic heterogeneity of primary human gliomas as it is important to describe markers for those gliomas that can be controlled by therapies that target glutamate metabolism and release. Health Relevance: We are using an innovative combination of techniques to characterize the complex changes in glutamate metabolism that we hypothesize occur in human gliomas and in an animal model. We anticipate that these data will help to develop new approaches to therapy and may also allow for the development of methods to rapidly generate a functional metabolic profile of individual gliomas that may lead to better, individualized treatment of these devastating tumors. PUBLIC HEALTH RELEVANCE: Brain tumors release the neurotransmitter glutamate in very high concentrations which can kill the surrounding tissue, allowing the tumor to grow within the confined space of the skull. The specific metabolic pathway used by these tumors to generate the released glutamate has not been determined and presents a possible therapeutic avenue. In this grant, we will expose samples of resected primary human brain tumors to a variety of stable isotopes of potential glutamate precursors to elucidate 1) the metabolic pathway by which gliomas make the glutamate that is released into the extracellular space;and 2) if different metabolic fuels can limit the synthesis and release of glutamate. Parallel studies in tumor-bearing mice will also be done and will allow us to determine if a low carbohydrate diet can alter glutamate metabolism and thus potentially tumor growth. Finally, as part of this proposal, we will identify metabolic biomarkers for those tumors that might respond to dietary therapy.